User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/17: Difference between revisions
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* Bacterial transformation , Long transformation protocol | * Bacterial transformation , Long transformation protocol | ||
** Add total volume (10.0 μL) to 50 μL BL21 and DH5α, Incubate on ice for 1 min., heat shock at 42°C for exactly 45 sec., immediately place on ice for 1 min. Add 800 μL sterile SOC medium. Grow with shaking at 37°C for 45 min. Pellet the cells for 3 min. at room temp. Resuspend the cells in 100 μL LB + AMP antibiotic. Plate cells on pre-warmed LB agar + AMP antibiotic. Grow overnight at 37°C. | ** Add total volume (10.0 μL) to 50 μL BL21 and DH5α, Incubate on ice for 1 min., heat shock at 42°C for exactly 45 sec., immediately place on ice for 1 min. Add 800 μL sterile SOC medium. Grow with shaking at 37°C for 45 min. Pellet the cells for 3 min. at room temp. Resuspend the cells in 100 μL LB + AMP antibiotic. Plate cells on pre-warmed LB agar + AMP antibiotic. Grow overnight at 37°C. | ||
== Result == | == Result == | ||
<font color="red">'''100s of colonies on plates with DH5α , No colonies on plates with BL21.''' | <font color="red">'''100s of colonies on plates with DH5α , No colonies on plates with BL21.''' |
Revision as of 13:59, 4 November 2013
PcTF Subcloning in E. coli | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Golden Gate Assembly of BD002 to Replace HsvtkTATA with CMV and HPK PromotersDesired Constructs: BD003: 5XGAL4-Spacer-CMV-Kozak-AMCyan-AmCyan-NLS-Stop BD004: 5XGAL4-Spacer-HPK-Kozak-AMCyan-AmCyan-NLS-Stop Existing construct is: 5XGAL4-Spacer-HsvTK-Kozak-AMCyan-AmCyan-NLS-Stop in V0200 mammalian vector Amplification of CMV and HPK from KAH187 and KAH184 respectively. The backbone vector has BSMBI cut site within the hygromycine resistance gene. I was removed though Site Directed Mutagenesis PCR Reactions
Backbone vector [math]\displaystyle{ X= (4600 / 209 ) * 0.013 * 20 = 5.72 }[/math] HPK [math]\displaystyle{ X= (2203 / 70 ) * 0.013 * 20 = 8.18 }[/math] CMV [math]\displaystyle{ X= (2275 / 42 ) * 0.013 * 20 = 14.08 }[/math]
Result100s of colonies on plates with DH5α , No colonies on plates with BL21. |